The purpose of this work is to determine the most physically effective radiation energy for K-edge absorption of x- or gamma-rays by iododeoxyuridine (IUdR) on Chinese hamster ovary (CHO) cells. Brachytherapy sources (Sm-145, I-125, Yb-169 and Am-241) and x-ray beams (30 kVp, 100 kVp and 100 kVp with gold, gadolinium, lead or tungsten filtration) were investigated for their preferential absorption qualities by IUdR sensitized DNA. The 30 kVp, 100 kVp and 100 kVp with tungsten filtration were then used to irradiate CHO cells, with or without IUdR incorporation (i.e. 10(-5) M of IUdR for 3 days). Radiation absorption calculations were performed to determine the increase in energy absorption in DNA with and without IUdR incorporated. In order to measure the in vitro biological effects of K-edge absorption, cell survival experiments were performed. The radiation physics calculations yielded an iodine dose enhancement ratio (DER) of 1.4+/-0.15. 1.8+/-0.15 and 2.7+/-0.15 for the 30 kVp, 100 kVp and tungsten filtered 100 kVp respectively, for 18% IUdR replacement of thymidine in DNA. The corresponding cell sensitization enhancement ratios (SER), determined from the cell survival assay, were determined to be 1.24+/-0.2, 1.8+/-0.2 and 2.3+/-0.3 for the 30 kVp, 100 kVp and tungsten filtered 100 kVp respectively, for cells with 18+/-2% IUdR incorporation. These SER values are in reasonable agreement with the DER values of 1.4, 1.8 and 2.7. From these radiation calculations and radiobiology experiments we confirm that using x-radiation energies above the K-edge of iodine (33.2 keV) can have a significant effect on cell survival. This effect is due mainly to the increase in the local dose to the DNA for IUdR-sensitized cells compared with the normal DNA which lacks the iodine contrast agent. Our results support the clinical application of IUdR and low-energy brachytherapy, perhaps using new technologies such as the x-ray needle or new isotopes such as Yb-169.

Human Cu,Zn superoxide dismutase (SOD) is a single domain all beta-sheet protein with its eight beta-strands arranged as a Greek key beta-barrel or immunoglobulin fold. Three circularly permuted variants of SOD were made by joining the native amino- and carboxy-termini, and introducing new termini at sites originally within connections between beta-strands. The locations of the new termini were chosen to interrupt beta-turns between the two N-terminal beta-hairpins and the short cross-barrel Greek key connection. Expression levels in the Escherichia coli periplasm were indistinguishable from that of native SOD. Reaction rates for the purified proteins were similar to those of the native enzyme, indicating that the permutants are correctly folded. Interrupting the covalent cross-bracing provided by the Greek key connection reduced the stability of the protein by approximately 1.0 kcal/mol, indicating only a slight contribution to conformational stability. The experiments test and eliminate two hypotheses for folding pathways for Greek key beta-barrels that require N-terminal beta-hairpins or covalent attachment across the short Greek key connection.

We investigated the DNA damage from Auger electrons emitted from incorporated stable iodine (127I), following photoelectric absorption of external x-rays. The effectiveness of the Auger electrons in producing DNA double-strand breaks (DSB) was determined theoretically, using Monte Carlo simulations of the radiation physics and chemistry, and was shown to be in reasonable agreement with DNA damage measured using the comet assay. The DSB yields were measured in CHO cells for 60Co (as a non-Auger-promoting radiation) and for tungsten-filtered 100 kVp x-rays capable of producing Auger electron emission. The theoretical study showed that on average, 2.5 Auger electrons were emitted for N-shell orbital vacancies and up to 10 Auger electrons were emitted from L1-shell vacancies. These Auger bursts produced approximately 0.03 DSB per N-shell vacancy and 0.3 DSB per K-shell or L-shell vacancy. The calculated yield of DSB from Auger cascades per unit dose (1 Gy) in water was approximately 1.7 for tungsten-filtered 100 kVp x-rays, assuming 20% IUdR substitution of thymidine. The comet assay yielded an experimental value of 3.6+/-1.6 per 1 Gy for the same conditions. The Monte Carlo simulations also demonstrated a high complexity of DSB produced by Auger cascades with virtually all DSB from inner shell orbitals (i.e. K, L shells) accompanied by compounded strand breakage and base damage, indicating a difficult lesion to repair. This finding agrees well with comet assay results of DNA repair, where an increase in the DSB yield in IUdR-sensitized cells was shown to persist after a time of 24 h. We conclude that Auger cascades in iodine produce a modest increase in the number of initial strand breaks of the order of 10% but the complex nature of these DSB makes them very difficult to repair or potentially prone to misrepair. The accentuated DNA damage may have major consequences for cell survival and may be exploitable in kilovoltage photon activation therapy (PAT) of tumors sensitized with iodine.

No abstract